JMJD6–BRD4 complex stimulates lncRNA HOTAIR transcription by binding to the promoter region of HOTAIR and induces radioresistance in liver cancer stem cells

Background Long non-coding RNA (lncRNA) HOTAIR acts importantly in liver cancer development, but its effect on radioresistance remains poorly understood. Here, our study probed into the possible impact of HOTAIR in radioresistance in liver cancer stem cells (LCSCs) and to elucidate its molecular basis. Methods Following sorting of stem and non-stem liver cancer cells, LCSCs were identified and subjected to RNA-seq analysis for selecting differentially expressed genes. Expression of HOTAIR was determined in liver cancer tissues and CSCs. The stemness, proliferation, apoptosis and radioresistance of LCSCs were then detected in response to altered expression of HOTAIR-LSD1-JMJD6-BRD4. Results Ectopic HOTAIR expression was found to promote radioresistance of LCSCs by maintaining its stemness. Mechanistic investigations indicated that HOTAIR recruited LSD1 to the MAPK1 promoter region and reduced the level of H3K9me2 in the promoter region, thus elevating ERK2 (MAPK1) expression. JMJD6–BRD4 complex promoted HOTAIR transcription by forming a complex and positively regulated ERK2 (MAPK1) expression, maintaining the stemness of LCSCs, and ultimately promoting their radioresistance in vitro and in vivo. Conclusion Collectively, our work highlights the promoting effect of the JMJD6–BRD4 complex on the radioresistance of LCSCs through a HOTAIR-dependent mechanism. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04394-y.


Introduction
Globally, the incidence of liver cancer continues to rapidly rise, and which has become the second leading reason contributing to cancer-related deaths [1][2][3].Currently, surgical excision, liver transplantation and interventional radiological treatment are the most effective therapies for the treatment of low-grade liver cancer [4,5].However, because of indistinguishable or delayed appearance of clinical symptoms, few patients could receive above-mentioned managements  [6].Notably, the development of radiotherapy techniques has shown satisfied local-regional control effect for tumor cells, offering a promising option for most patients with advanced liver cancer [7].But radioresistance acquisition remains a serious obstacle for the successful treatment of liver cancer patients [8].Thus, there is an urgent need to explore potential targets for overcoming radiotherapy resistance and further improving radiotherapy efficacy in the clinical settings.
Accumulating studies have demonstrated that cancer stem cells (CSCs), a distinct sub-population of cells, exhibit unlimited self-renewal potential, exclusive tumorigenicity, and resistance to chemo/radiotherapy [9][10][11].LCSCs in liver cancer tissues may lead to resistance and metastasis, while standard managements cannot eradicate the resistant LCSCs [12,13].Hence, it is essential to reveal the underlying molecular mechanisms linked to LCSCs for the diagnosis and treatment of liver cancer [14].CD13 and CD133 are generally recognized surface markers of LCSCs [15,16], and based on the analysis of differentially expressed genes of CD13 and CD133-positive cell subgroups, we found that long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is up-regulated in LCSCs.LncRNAs are non-coding RNAs consisting of more than 200 nucleotides in length [17].LncRNAs compete with microRNAs (miRNAs) for binding and can be processed into miRNAs [18], and specifically, lncRNAs are implicated in diverse biological activities and bears great responsibility for modulation of diseases and cancers [19,20].HOTAIR, located in the Homeobox C gene cluster, is a lncRNA with regulatory functions of transcription and has been demonstrated to mediate gene expression through epigenetic modifications, like chromatin modifications [21] and DNA methylation [22,23].Multiple studies have indicated that overexpression of HOTAIR in liver cancer is positively correlated to poor prognosis, recurrence and tumor progression [24,25].A recent study has shown that HOTAIR regulates myeloid differentiation through the upregulation of p21 via miR-17-5p in acute myeloid leukaemia [26].In addition, HOTAIR engages in myocardial regeneration in neonatal mice [27] and HOTAIR is capable of facilitating the activation and fibrosis of stellate cells in the liver [28].Moreover, HOTAIR can regulate the development of leukemia by modulation of DNA and histone methylation [29].Furthermore, HOTAIR has been highlighted to interact with lysine-specific demethylase 1 (LSD1) [30], and LSD1 expression is enhanced in liver cancer cells [31].Therefore, in the nucleus, HOTAIR tethers protein complexes to chromatin to repress or activate gene transcription, thus controlling the progression of liver cancer.
In addition, it is interesting to know that jumonji domain containing 6 (JMJD6) and bromodomain-containing protein 4 (BRD4) can be recruited to the HOTAIR promoter region to promote HOTAIR expression in breast cancer and glioblastoma multiforme [32,33], and the JMJD6-BRD4 complex can modulate the expression of genes [34].Furthermore, elevated expression of JMJD6 can maintain cancer stemness properties [35], and SKLB325, an inhibitor of JMJD6, can effectively impede the progression of cancer [36].Herein, from the abovementioned findings, we hypothesize that the JMJD6-BRD4 complex may mediate HOTAIR expression, which in turn affects the radioresistance of liver cancer by interacting with LSD1.To address this hypothesis, we studied the correlation between JMJD6-BRD4 complex and HOTAIR expression in clinical tissue samples of liver cancer patients, LCSCs and xenograft tumor in nude mice, as well as the effect of their interactions on the radioresistance of LCSCs, aiming at clarifying the mechanistic actions of HOTAIR and providing a novel therapeutic target for liver cancer.

Ethics statement
The clinical research was approved by the Ethics Committee of Xuzhou Cancer Hospital and conducted in accordance with the Declaration of Helsinki.All participants signed informed consent documentation before sample collection.

Study subjects
This study enrolled 50 patients (aged 31 to 68 years) diagnosed with liver cancer by aspiration biopsy and pathological examination at Xuzhou Cancer Hospital from September 2017 to February 2019.The diagnosis of liver cancer was carried out according to the standards of the World Health Organization.All the tumor samples were confirmed to contain more than 80% tumor cells through pathological examination.Tissue samples were obtained from patients who did not receive any anti-cancer treatment.Pathological examination was performed to confirm that all tumor nodules were completely resected by removing tumor-free tissue on the liver surface.Complete clinical pathology and follow-up data were collected, and the death cases from non-liver cancer or accidents were excluded.

Immunohistochemistry
Paraffin-embedded and sectioned clinical tissue specimens were dewaxed to water, dehydrated in an alcohol gradient, washed twice in distilled water, treated with 3% methanolic hydrogen peroxide for 20 min, washed in distilled water for 2 min, and 3 min in 0.1 M PBS.

Radiotherapy treatment
The Astrophysics Torrex X-ray Inspection System (Model 120D, Scanray Corporation) was used to irradiate cells with X-ray at 115 kVp and 5 mA.The cells in the logarithmic growth phase were seeded in DMEM containing 10% FBS, and 100 U/mL penicillin-streptomycin at 1 × 10 5 cells/mL, and cultured in a 5% CO 2 incubator at 37 °C.If transfection was needed, the cells were irradiated 24 h after transfection with 6 Gy (dose rate: 0.341 Gy/min, radiation area: 10 cm × 10 cm) of X-ray, and continued to be cultured for 24 h for subsequent experiments.

Microsphere formation assay
The standard medium was made by serum-free DMEM/ F12 (1:1) basic medium, the hepatocyte growth factor (HGF) (10 ng/mL), the recombinant growth factor (bEGF) (20 μg/mL) and B27.The cells to be tested were cultured with 2 mL standard medium at a low-adhesion six-well plate in the incubator (Thermo Fisher Scientific, Inc., Waltham, MA) with 37 °C and 5% CO 2 .Two weeks later, the images of cells were obtained under a microscope and the number of microspheres with volume greater than 100 μm was counted.

Clonogenic assay
The cells to be tested were seeded into 6-well plates, and exposed to X-ray (6 Gy) upon reaching the density of approximately 50%.After 7 days, the culture medium was discarded, and the cells were fixed with methanol for 10 min.Next, the cells were stained with crystal violet, and the results were recorded by photos.More than 50 cell clones were counted under an ordinary inverted microscope, and then the colony formation rate was calculated as follows: colony formation rate (%) = (number of colonies/ number of inoculated cells) × 100%.

Quantification of gene expression
RT-qPCR was adopted to measure the mRNA expression of target genes.According to the instructions, TRIzol kit (Invitrogen, Carlsbad, CA) was employed to extract total RNA from tissues or cells and measured RNA concentration.The primers used in this study were synthesized by Dalian Takara Biotechnology Co., Ltd.(Dalian, Liaoning, China) (Additional file 1: Table S2).Then RT-qPCR assay was conducted by complementary DNA reverse expression kit (K1622, Beijing Yaanda Biotechnology Co., Ltd., Beijing, China) and fluorescence quantitative PCR system (ViiA 7, Da An gene Co., Ltd. of Sun Yat-sen university, Guangzhou, Guangdong, China).The expression of genes was normalized to GAPDH, and the results were calculated by 2 −△△CT method.

RIP assay
According to the manufacturer's instructions, LSD1 antibody (1:50, #2139, Cell Signaling Technology), BRD4 (1:100, #83375, Cell Signaling Technology), and Magna RIP ™ RIP kit (Millipore, Bedford, Massachusetts) were used for RIP assay.RNA was isolated from the immunoprecipitation product of the cells and quantified by a nanophotometer spectrophotometer (Implen Applications Note, Munich, Germany) of UV-visible light spectra.Enrichment of HOTAIR or LSD1 was detected by RT-qPCR.

Cellular tumorigenicity in nude mice
Hep3B cells (6 × 10 6 cells) were obtained and resuspended in 50 μL mixture (PBS: Matrigel = 1:1).Thirty nude mice, aged 5-6 weeks, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.(Shanghai China) and subcutaneously injected with above-mentioned cells.After 10 days, the injection site was treated with X-ray irradiation (10 Gy).These nude mice were equally divided into 5 groups (n = 6): shCtl + oe-NC group, shJMJD6 + oe-NC group, shJMJD6 + oe-ERK2 group, PBS group and SKLB325 (JMJD6 inhibitor) group.After tumor cell injection for 7 days, the mice were intraperitoneally injected with PBS or SKLB325 once a week for 4 consecutive weeks.Following this, the mice were euthanized, and the tumor tissues were excised, measured and photographed.

TUNEL assay
The procedures before hydration were the same as the immunohistochemistry process.Next, slices were immersed in 0.85% NaCl and 4% paraformaldehyde in sequence, and then exposed to 20 μg/mL proteinase K solution.After treatment with 4% paraformaldehyde, the slices were incubated with rTdT reaction solution and then stained with 2× SSC solution.Thereafter, the slices were incubated with HRP and added with DAB for color reaction.Following sealing with neutral resin, the slices were observed under a microscope, with the positive rate calculated.

RNA-seq analysis
Illumina NextSeqCN500 with Ribo-Zero protocol (Illumina, Inc., based in San Diego, CA) can be used to generate HiSeqLibrary with 950 ng mRNA.RNA sequencing (Illumina HiSeq) was performed on CD13 + CD133 + LCSC subsets and negative liver cancer cell subsets.Next, Illumina's RTA (v1.18) software was used for image analysis and base detection.The sequencing library was generated using NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB) according to the manufacturer's recommendations, and the index code was added to the attribute sequence of each sample.Demultiplexing and FASTQ format file generation and alignment were performed using HiSeq analysis software (TopHat v2.1.1)and UCSC human genome version mm10.Thereafter, RNA-SeQC v1.1.8was used for quality control, and HTSeq-counts v0.7.2 was used to generate counts.After converting the count data into FPKM (Fregments Per Kilobase per Million) format data, the R "limma" software package was applied to determine the differentially expressed genes between the CD13 + CD133 + LCSC subsets and the negative liver cancer cell subsets with |logFC|> 1 and p.value < 0.05 as cut-off values.

In silico analysis
For RNA-seq data, TCGA database was used to obtain the RNA-seq data in the FPKM format in the liver cancer project, which were then converted into the FPKM format into transcripts per million reads format and subjected to log2 conversion.The Wilcoxon rank sum test was adopted to calculate the difference in genes between the normal and tumor groups.A value of p < 0.05 was considered to be indicative of statistical significance.The clinical data of liver cancer patients were obtained from TCGA database.The "survminer" package (version 0.4.9)(for visualization) and "survival package" (version 3.2-10) (for statistical analysis of survival data) were employed to analyze the correlation between the expression of LSD1 and ERK2 (MAPK1) and the overall survival of liver cancer patients.The expression grouping was set to 0-50 vs 50-100, and the statistical method was Log-rank test.A value of p < 0.05 was considered to be indicative of statistical significance.

Statistical analysis
The statistical analyses were performed by SPSS 21.0 (IBM Corp., Armonk, NY), and the measurement data were expressed as mean ± standard deviation.Data obeying normal distribution and homogeneity of variance between cancer tissues and adjacent normal tissues were compared by paired t test, and those between the other two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way analysis of variance (ANOVA) with Tukey's post-hoc test.Kaplan-Meier method was employed to evaluate the survival of patients, and log-rank test was used for univariate analysis.The results were considered statistically significant when p < 0.05.

HOTAIR is upregulated in liver cancer tissues and LCSCs, which correlates to stemness maintenance and radioresistance of LCSCs
It has been recognized that CD13 and CD133 are surface markers of LCSCs [16].Through flow cytometric sorting, the CD13 + CD133 + liver cancer cell subsets in Hep3B and Huh7 cells were enriched and respectively defined as Hep3B and Huh7 CSCs.Analysis of differentially expressed genes in CD13 + CD133 + or negative liver cancer cell subsets in the RNA-sequencing (RNA-seq) data suggested that HOTAIR was highly expressed in LCSCs, with significant difference (Fig. 1A).The differential gene heat map is shown in Additional file 2: Figure S1.In addition, the lncDiseas database showed that HOTAIR was related to hepatocellular carcinoma diseases (Additional file 1: Table S3).Furthermore, analysis using TCGA database indicated that HOTAIR was abundantly expressed in cancer tissues of patients with liver cancer (Fig. 1B) (p = 0.03) and that the expression of HOTAIR was negatively correlated with the progression free survival of patients with liver cancer (Fig. 1C).As illustrated by RT-qPCR, HOTAIR expression was higher in liver cancer tissues than that in adjacent normal tissues (Fig. 1D).
RT-qPCR results confirmed the silencing and overexpression efficiency of HOTAIR in Hep3B and Huh7 CSCs (Fig. 1E).The microsphere formation assay data displayed that HOTAIR silencing reduced the stemness of LCSCs, while overexpression of HOTAIR led to reversed condition (Fig. 1F, Additional file 2: Figure S2A).Meanwhile, HOTAIR silencing facilitated the inhibitory effect of X-ray irradiation on the colony formation ability of LCSCs, while opposite trend was witnessed upon overexpression of HOTAIR (Fig. 1G, Additional file 2: Figure S2B).The aforementioned results indicated that HOTAIR was overexpressed in liver cancer tissues and LCSCs.This overexpression was related to stemness maintenance and radioresistance of LCSCs.

LSD1 interacts with HOTAIR to maintain the stemness of LCSCs and enhance their radioresistance
LSD1 has been reported to interact with HOTAIR in cancer cells [30], and the expression of LSD1 is enhanced in liver cancer cells [31].Similar to HOTAIR, LSD1 was found to be overexpressed in clinical samples of patients with liver cancer, positively linked to the malignancy degree (Fig. 2A).In addition, LSD1 expression was negatively linked to the overall survival of liver cancer patients (Fig. 2B).Moreover, LSD1 expression was much higher in liver cancer tissues (Fig. 2C).
To analyze the role of LSD1 in the stemness maintenance of LCSCs and their radioresistance, we silenced LSD1 in Huh7 and Hep3B CSCs.The results of Western blot presented that both shLSD1#1 and shLSD1#2 sequences effectively decreased the expression of LSD1, with shLSD1#2 showing the better silencing efficiency and was selected for following experimentations; and overexpression efficiency of LSD1 was also confirmed (Fig. 2D).The percentage of cells that formed microspheres was measured in Hep3B and Huh7 CSCs with LSD1 depletion, and the results showed that LSD1 depletion reduced the ability of LCSCs to form microspheres, while overexpression of LSD1 led to contrary finding (Fig. 2E, Additional file 2: Figure S2C), revealing critical role of LSD1 in the maintenance of LCSCs.In addition, the colony formation ability of LCSCs was inhibited in response to LSD1 silencing and X-ray exposure, while opposing tendency was detected upon overexpression of LSD1 (Fig. 2F, Additional file 2: Figure S2D).Furthermore, the results of RIP assay revealed that HOTAIR interacted with LSD1 (Fig. 2G).These results uncovered that LSD1 could interact with HOTAIR and that LSD1 maintained the stemness of LCSCs and thereby reduced the sensitivity of LCSCs to radiotherapy.

HOTAIR promotes extracellular signal-regulated kinase 2 (ERK2) expression by recruiting LSD1 to the promoter region of MAPK1
Previous reports have revealed that HOTAIR has been recognized as a scaffold molecule of LSD1 to modulate the expression of genes [30,38], and LSD1 could further promote ERK expression [38].More importantly, The experimental data were summarized as mean ± standard deviation.Data between cancer tissues and adjacent normal tissues were compared by paired t test, and those between the other two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test.Cellular experiments were repeated three times abnormal activation of ERK could enhance radioresistance of liver cancer cells [39].Therefore, we hypothesized that HOTAIR might act as a scaffold molecule to recruit LSD1 to the MAPK1 promoter region, further mediate ERK2 (MAPK1) expression, and ultimately increase radioresistance of liver cancer.For validation, we first detected the expression of ERK2 (MAPK1) in Hep3B and Huh7 CSCs by RT-qPCR and Western blot, the results of which showed that the protein and mRNA levels of ERK2 (MAPK1) were diminished in Hep3B and Huh7 CSCs after LSD1 depletion or HOTAIR depletion (Fig. 3A, B).Furthermore, LSD1 overexpression could up-regulate ERK2 (MAPK1) expression, while simultaneous treatment of shHOTAIR and overexpression (oe)-LSD1 in Hep3B CSCs didn't cause significant changes in the ERK2 (MAPK1) expression (Fig. 3C, D), implying that the effect of LSD1 on ERK2 (MAPK1) depended on HOTAIR.Meanwhile, the results of ChIP assay suggested that in the presence of HOTAIR depletion, the binding of LSD1 to the MAPK1 promoter region was attenuated and the H3K9me2 enrichment was increased (Fig. 3E).
The above data demonstrated that HOTAIR could recruit LSD1 to the promoter region of MAPK1 and then trigger the demethylation of H3K9me2 in the MAPK1 promoter, thus modulating ERK2 (MAPK1) expression.

LSD1 enhances ERK2 (MAPK1) expression, thus maintaining the stemness of LCSCs and enhancing their radioresistance
We moved to investigate whether the effect of HOTAIR-LSD1 axis on increasing radioresistance was achieved by regulating ERK2 (MAPK1).Based on TCGA database, we analyzed the ERK2 (MAPK1) expression in liver cancer and its relationship with the overall survival of liver cancer patients, and the results revealed that ERK2 (MAPK1) was highly expressed in clinical tissue samples of liver cancer patients (Fig. 4A) and that ERK2 (MAPK1) expression was negatively correlated with the overall survival of patients (Fig. 4B, Additional file 2: Figure S3A).Moreover, the results of immunohistochemistry demonstrated that the positive expression of LSD1 and ERK2 (MAPK1) was increased in liver cancer tissues (Fig. 4C).Next, we designed two independent shRNAs targeting ERK2 (MAPK1) (shERK2#1 and shERK2#2) and transfected them into Hep3B and Huh7 CSCs.The two shR-NAs had excellent silencing efficiency, but shERK2#2 (written as shERK2 in the following text) showed a superior silencing efficiency (Fig. 4D) and was chosen for subsequent experiments.Interestingly, results have revealed that ERK2 (MAPK1) activation was critical for maintaining the stemness of stem cells [40].Here, in the presence of ERK2 (MAPK1) silencing, the microsphere formation ability of Hep3B and Huh7 CSCs was reduced (Fig. 4E), revealing the significance of ERK2 (MAPK1) in maintenance of LCSCs subsets.Meanwhile, ERK2 (MAPK1) silencing accelerated the inhibitory effect of X-ray on the colony formation ability of LCSCs (Fig. 4F).Western blot results revealed that silencing of LSD1 decreased ERK2 (MAPK1) expression, which was reversed after further ERK2 (MAPK1) overexpression (Fig. 4G, Additional file 2: Figure S3B).In addition, silencing of LSD1 was found to attenuate the ability of LCSCs to form microspheres, but restored ERK2 (MAPK1) expression reversed this result (Fig. 4H).After treatment with X-ray irradiation, LSD1 silencing markedly restricted the proliferation of LCSCs, and ERK2 (MAPK1) overexpression reversed above effects (Fig. 4I).Overall, these results suggested that LSD1 could upregulate ERK2 (MAPK1) expression to maintain the stemness of LCSCs and enhance their radioresistance.

JMJD6 maintains the stemness of LCSCs and enhances their radioresistance by promoting HOTAIR expression
As previously reported, JMJD6 and BRD4 could be recruited to the HOTAIR promoter region and further increase HOTAIR expression [32,33].Besides, JMJD6 and BRD4 could form a complex to modulate gene expression [34], so we speculated that the JMJD6 may form a complex with BRD4 to regulate the expression of HOTAIR.
For verification, we first detected the expression of JMJD6 and BRD4 in liver cancer tissues and adjacent normal tissues by RT-qPCR.As shown in Fig. 5A, BRD4 and JMJD6 were highly expressed in liver cancer tissues.Additionally, the expression of HOTAIR and ERK2 (MAPK1) was diminished in Hep3B and Huh7 CSCs after JMJD6 silencing or BRD4 silencing (Fig. 5B, Additional file 2: Figure S4).Next, we analyzed the effect of JMJD6 or BRD4 on microsphere forming ability of LCSCs, and the results uncovered that JMJD6 silencing or BRD4 silencing reduced microsphere forming ability (Fig. 5C,  D).Further, the results of ChIP assay on LCSCs displayed that JMJD6 silencing reduced the enrichment of JMJD6 and BRD4 in the HOTAIR promoter region (Fig. 5E).The above results indicated that the JMJD6-BRD4 complex enhanced HOTAIR expression by binding to its promoter region.
We first detected the expression of LSD1 in JMJD6silenced cells, and the results depicted that there was no significant change in LSD1 expression after JMJD6 silencing (Fig. 5F, Additional file 2: Figure S3C), suggesting that although JMJD6 regulated HOTAIR transcription by binding to its promoter region, and regulated the binding of LSD1 in the ERK2 promoter region and the enrichment of H3K9me2 through HOTAIR, which affects the radioresistance of LCSCs, but HOTAIR did not affect the expression of LSD1 in cells.Next, we overexpressed HOTAIR in JMJD6 silencing cells to the base level, and illustrated that the expression of ERK2 (MAPK1) was impeded in response to JMJD6 depletion, while HOTAIR overexpression negated this effect (Fig. 5G).Consistently, an obvious decline in cell microsphere formation and colony formation ability caused by JMJD6 silencing could be reversed by further overexpression of HOTAIR (Fig. 5H, I).It has been reported that JMJD6 can catalyze the hydroxylation of lysine residues, and 19 hydroxylation sites located in the lysine-rich region on BRD4 can be modified by it [41].As proved by RIP, BRD4 could interact with LSD1 (Fig. 5J).In summary, the JMJD6/ BRD4 complex exerted a direct effect on LSD1.
These data revealed that the JMJD6-BRD4 complex could bind to the HOTAIR promoter to elevate HOTAIR expression and promote ERK2 (MAPK1) expression, maintaining the stemness of LCSCs and enhancing cell radioresistance.

The role of JMJD6 in promoting radioresistance of LCSCs depends on the transcriptional regulation of ERK2 (MAPK1) by HOTAIR-LSD1 axis
Further, through cellular tumorigenicity in nude mice, we explored whether JMJD6-mediated radioresistance in liver cancer cells was related to the regulation of ERK2 (MAPK1) by the HOTAIR-LSD1 axis.Hep3B CSCs with JMJD6 silencing or in combination with ERK2 (MAPK1) overexpression were subcutaneously injected to nude mice, followed by exposure to X-ray radiation.After treatment with X-ray, the tumor-forming ability of cells in nude mice was markedly diminished in response to JMJD6 depletion, while further ERK2 (MAPK1) overexpression increased the tumor volume and recovered the resistance of cells to radiation (Fig. 6A, B).
Moreover, in the tumor tissues of mice after JMJD6 silencing, the expression of HOTAIR was restricted, while further overexpression of ERK2 (MAPK1) did not affect the expression of HOTAIR (Fig. 6C).The results of immunohistochemistry showed that JMJD6 depletion alone could impede the positive expression of ERK2 (MAPK1), which was negated after both JMJD6 depletion and ERK2 (MAPK1) overexpression (Fig. 6D).More importantly, the results of Ki-67 staining and TUNEL assay indicated that silencing of JMJD6 inhibited the tumor radioresistance and enhanced cell apoptosis.However, ERK2 (MAPK1) overexpression negated the impact of JMJD6 silencing (Fig. 6E, F).To sum up, the promoting effect of JMJD6 on radioresistance of LCSCs may rely on the transcriptional regulation of ERK2 (MAPK1) by HOTAIR-LSD1 axis.

JMJD6 inhibitor SKLB325 reduces radioresistance of LCSCs by repressing LCSC stemness
The first JMJD6 inhibitor SKLB325 and its role in cancer were reported in 2019, and until now, few studies focused on the application of JMJD6 inhibitor in cancer [36].Therefore, on the basis of above study, we further investigated the effect of JMJD6 inhibitor on cancer.As presented in Fig. 7A-C, SKLB325 restricted the expression of HOTAIR, MAPK1 and stem cell marker genes Sox2 and Oct2 in Hep3B and Huh7 CSCs.Besides, after treatment with SKLB325, the percentage microsphere formation in LCSCs was reduced (Fig. 7D), indicating that SKLB325 could effectively restrict the stemness of LCSCs.Meanwhile, SKLB325 accelerated the inhibitory effect of X-ray on the colony formation ability of LCSCs (Fig. 7E).In the nude mice with xenograft tumors, the tumor-forming ability of SKLB325-treated LCSCs was markedly reduced after radiation (Fig. 7F, G).In addition, after SKLB325 treatment, the expression of HOTAIR and ERK2 (MAPK1) was diminished in the tumor tissues (Fig. 7H, I), accompanied with slow proliferation and accelerating apoptosis of tumor cells (Fig. 7J).Collectively, JMJD6 inhibitor SKLB325 could effectively attenuate radioresistance of liver cancer cells by reducing LCSC stemness in vivo and in vitro.

Discussion
Radiotherapy is a more and more important tool in for the treatment of primary and metastatic liver tumors, and low-dose radiation could restrict unresectable primary tumors or liver metastases [42].However, although the efficacy of this therapy has been improved in recent years, ultimately radiation delivery was impeded by radioresistance, high toxicity, and a relatively high occurrence of liver failure [8,43].According to bioinformatics analysis, we uncovered that HOTAIR was expressed at high levels in liver cancer tissues, and its expression has also been reported to be closely associated with the development and poor prognosis of liver cancer [24,25].But few studies focus on the effect of HOTAIR on the radioresistance of liver cancer cells, as well as its related molecular mechanisms.In the present study, we demonstrate that HOTAIR maintains the LCSC stemness and further enhances their radioresistance the JMJD6-BRD4-HOTAIR-LSD1-ERK2 (MAPK1) axis (Fig. 8).
HOTAIR plays a pivotal role in the pathogenesis of several cancers, the ectopic expression of which is involved in drug resistance, enhancing cell viability and metastasis through various signaling pathways in tumors [44].Hence, the properties of HOTAIR have aroused interest of using HOTAIR as a potential therapeutic target or biomarker in cancer biology [45,46].In addition, the increased expression of HOTAIR is highly linked to cell metastasis, drug tolerance and unfavorable overall survival of patients [47].In the present study, we also implied that HOTAIR enhanced the radioresistance by maintaining the stemness of LCSCs.
As highly reported, HOTAIR could act as an LSD1 scaffold molecule to modulate the expression of genes [30,48,49].Moreover, LSD1 expression has been found to be enhanced in several cancers and this enhancement can facilitate the proliferation, drug resistance and transformation of cancer cells in vitro and in vivo [50][51][52].Liu et al. has uncovered that LSD1 is highly expressed in LCSCs and closely linked to liver cancer progression [33], which is consistent with our results in Fig. 2. Further, Li et al. have illustrated that LSD1 up-regulates the expression of ERK [38].ERK plays a significant role in the intracellular regulating network, and the MAPK-ERK cascades can be activated by various stimuli, including the extracellular matrix and growth factors [53][54][55].Besides, CD133 + LCSCs display a marked increase in the MAPK pathway activation, as supported by phosphorylated ERK level [56].Meanwhile, ERK signal has been proposed to be implicated in the progression of various human tumors by mediating the expression of genes, such as cyclin D1 [57] and Bax [58].In this study, we demonstrated that HOTAIR maintained the LCSC stemness and enhance its radioresistance by regulating LSD1 and further promoting ERK2 (MAPK1) expression.
Consistent with our results, Piao et al. also uncovered that abnormal activation of ERK could increase the radioresistance of liver cancer cells [39].In addition, overexpression of HOTAIR has been reported to accelerate dyskinesia and facilitate dopaminergic neuron apoptosis in a Parkinson's disease mouse model via activation of the ERK1/2 axis [59].Another study has shown that HOTAIR depletion can promote apoptosis of lymphoma cells via inactivation of the ERK signaling pathway [60].
JMJD6, an epigenetic mediator of multilevel activities [35], has been unveiled to promote the expression of HOTAIR [32].Additionally, JMJD6 has been indicated to participate in the development of multiple cancers, including liver cancer [61][62][63], and the enzymatic activity of JMJD6 was pivotal for cancer progression [64,65].Thus, inhibiting JMJD6 activity was recognized as a promising solution for the treatment of JMJD6-involved cancers [66].Recently, Zheng et al. found a JMJD6 inhibitor, SKLB325, diminished intraperitoneal tumor weight and prolong the overall survival of tumor-bearing nude mice, implying that SKLB325 might be a potential candidate drug for cancer management [36].In the current study, our results also showed that SKLB325 restricted the stemness of LCSCs and attenuated their radioresistance in vivo and in vitro by mediating HOTAIR expression.
Taken together, our experimental results showed that HOTAIR impeded the radiosensitivity of liver Fig. 8 Mechanism graph of the regulatory network.The JMJD6-BRD4 complex elevates the expression of ERK2 through the HOTAIR-LSD1 axis, thereby maintaining the stemness of LCSCs and enhancing their radioresistance cancer cells by maintaining its stemness.Besides, the effect of HOTAIR on LCSCs was achieved by recruiting LSD1 to enhance ERK2 (MAPK1) expression.Further, the expression of HOTAIR was regulated by JMJD6-BRD4 complex, and the JMJD6 inhibitor contributed to the radiosensitivity of liver cancer cells by suppressing the expression of HOTAIR and ERK2 (MAPK1).Based on the data of our study, we demonstrated that JMJD6-BRD4 complex regulated the LCSC irradiation tolerance via the HOTAIR-LSD1-ERK2 (MAPK1) axis, which provided promising targets or biomarkers for the management of radioresistance in liver cancer.

Fig. 1 Fig. 2 LSD1
Fig.1HOTAIR expression is increased in liver cancer tissues and LCSCs, which links to stemness maintenance and radioresistance of LCSCs.A A Volcano map of the gene expression between CD13+CD133+ liver cancer cell subsets and negative liver cancer cell subsets based on the RNA-seq data.Red indicates highly expressed genes while green indicates poorly expressed genes.B The expression of HOTAIR in liver cancer and normal tissue samples in TCGA database (p = 0.03).C Correlation between the expression of HOTAIR and the progression free survival of patients with liver cancer.D The expression of HOTAIR in normal and liver cancer tissues measured by RT-qPCR, normalized to GAPDH.*p < 0.05 compared with adjacent normal tissues.E Silencing and overexpression efficiency of HOTAIR determined by RT-qPCR in Hep3B and Huh7 CSCs.*p < 0.05 compared with Hep3B and Huh7 CSCs treated with shCtl, # p < 0.05 compared with Hep3B and Huh7 CSCs treated with oeCtrl.F The effect of HOTAIR silencing or overexpression on the stemness maintenance of LCSCs, as detected by microsphere formation assay.G The colony formation ability of LCSCs after 6 Gy X-ray irradiation after HOTAIR silencing or overexpression, as examined by clonogenic assay.*p < 0.05 compared with Hep3B and Huh7 CSCs treated with shCtl, # p < 0.05 compared with Hep3B and Huh7 CSCs treated with oeCtrl.Data were represented as mean ± standard deviation.Data between cancer tissues and adjacent normal tissues were compared by paired t test, and those between the other two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test.Cellular experiments were repeated in triplicate

Fig. 3
Fig. 3 HOTAIR increases ERK2 (MAPK1) expression by recruiting LSD1 to the promoter region of MAPK1.A ERK2 (MAPK1) and LSD1 expression in Hep3B and Huh7 CSCs with LSD1 silencing or HOTAIR silencing as examined by Western blot, normalized to GAPDH.B MAPK1 and HOTAIR expression in Hep3B and Huh7 CSCs with LSD1 silencing or HOTAIR silencing examined by RT-qPCR, normalized to GAPDH.*p < 0.05 compared with Hep3B and Huh7 CSCs treated with shCtl.C The expression of HOTAIR in Hep3B CSCs treated with shHOTAIR or combined with oe-LSD1 measured by RT-qPCR, normalized to GAPDH.*p < 0.05 compared with shCtl + oe-NC-treated Hep3B CSCs.D Western blot assay of ERK2 (MAPK1) and LSD1 proteins in Hep3B CSCs treated with shHOTAIR or combined with oe-LSD1, normalized to GAPDH.E The relative enrichment of LSD1 and H3K9me2 in the promoter region of MAPK1 in Hep3B CSCs with HOTAIR silencing assessed by ChIP, relative to Input.*p < 0.05 compared with shCtl-treated Hep3B CSCs.The experimental data were represented as mean ± standard deviation.Results between two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test.Cellular experiments were repeated for three times

Fig. 4
Fig. 4 LSD1 increased ERK2 (MAPK1) expression, and thus maintained LCSCs stemness and further enhanced their radioresistance.A ERK2 (MAPK1) expression in normal samples and liver cancer samples, as analyzed by TCGA database.LIHC, liver hepatocellular carcinoma.Red box plots represent tumor samples, and gray box plots represent normal samples.*p < 0.01 compared with normal samples.B The correlation between ERK2 (MAPK1) expression and overall survival rate of liver cancer patients analyzed by TCGA database.The upper right is the significant p value and legend.C The positive expression of LSD1 and ERK2 (MAPK1) proteins in clinical liver cancer tissue samples, as measured by immunohistochemistry. *p < 0.05 compared with adjacent normal tissues.D The silencing efficiency of ERK2 (MAPK1) shRNAs in Hep3B and Huh7 CSCs, as detected by Western blot, normalized to GAPDH.E Microsphere formation assay was adopted to measure the stemness of LCSCs with ERK2 (MAPK1) silencing.*p < 0.05 compared with shCtl-treated cells.F The colony formation ability of LCSCs with ERK2 (MAPK1) silencing after exposure to 6 Gy X-ray, as measured by clonogenic assay.*p < 0.05 compared with shCtl-treated cells.G Quantitative analysis the relative expression of LSD1 and ERK2 (MAPK1) proteins in Hep3B and Huh7 CSCs in response to shLSD1, oe-ERK2 or in combination by Western blot, relative to GAPDH.H The LCSC stemness after treatment with shLSD1, oe-ERK2 or in combination measured by microsphere formation assay.I, After 6 Gy X-ray treatment, cell proliferation in the presence of shLSD1, oe-ERK2 or in combination, as examined by clonogenic assay.*p < 0.05 compared with shCtl + oe-NC-treated cells.#p < 0.05 compared with shLSD1 + oe-NC-treated cells.Above data were shown as mean ± standard deviation.Results between two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test.Cellular experiments were repeated in triplicate

Fig. 5
Fig. 5 JMJD6 enhances HOTAIR expression to maintain the stemness of LCSCs and promotes their radioresistance.A Quantitative analysis of JMJD6 and BRD4 expression in liver cancer and adjacent normal tissues measured by immunohistochemistry. *p < 0.05 compared with adjacent normal tissues.B The expression of JMJD6, BRD4 and ERK2 (MAPK1) in Hep3B and Huh7 CSCs with JMJD6 silencing or BRD4 silencing detected by Western blot, relative to GAPDH.*p < 0.05 compared with shCtl-treated cells.C The stemness of LCSCs with JMJD6 silencing or BRD4 silencing detected by microsphere formation assay.*p < 0.05 compared with shCtl-treated cells.D After exposure to X-ray, the cell colony formation in the presence of shJMJD6 or shBRD4, *p < 0.05 compared with shCtl-treated cells.E The enrichment of JMJD6 and BRD4 in the promoter region of HOTAIR after JMJD6 silencing, as examined by ChIP assay, relative to Input.The relative enrichment on the Y-axis represents JMJD6 or BRD4/IgG.*p < 0.05 compared with shCtl-treated cells.F Detection of expression of JMJD6 and LSD1 in cells following silencing of JMJD6, *p < 0.05 compared with shCtl-treated cells.G The expression of ERK2 (MAPK1) and JMJD6 after JMJD6 depletion or in combination with HOTAIR overexpression, as examined by Western blot and RT-qPCR.*p < 0.05 compared with shCtl + oe-NC-treated cells.#p < 0.05 compared with shJMJD6 + oe-NC-treated cells.H The microsphere formation assay was adopted to measure LCSC stemness after JMJD6 depletion or in combination with HOTAIR overexpression.*p < 0.05 compared with shCtl + oe-NC-treated cells.#p < 0.05 compared with shJMJD6 + oe-NC-treated cells.I Cell colony formation after JMJD6 depletion or in combination with HOTAIR overexpression following 6 Gy X-ray treatment examined by clonogenic assay.*p < 0.05 compared with shCtl + oe-NC-treated cells.#p < 0.05 compared with shJMJD6 + oe-NC-treated cells.J, RIP assay validated the interaction of JMJD6/ BRD4 complex with LSD1 (relative enrichment in Y-axis represents IP-LSD1/IgG, * p < 0.05 compared with IgG group.Measurement data were expressed as mean ± standard deviation.Results between two groups were compared by unpaired t test.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test.Cellular experiments were repeated three times

Fig. 6 JMJD6
Fig. 6 JMJD6 promotes radioresistance of LCSCs by regulating the HOTAIR-LSD1-ERK2 axis.A The diameter of xenograft tumors of nude mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression (n = 6).B The weight of xenografted tumors in nude mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression (n = 6).C The expression of HOTAIR in xenografted tumor tissues of nude mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression determined by RT-qPCR, normalized to GAPDH (n = 6).D The ERK2 (MAPK1) positive cells in xenograft tumor tissues of nude mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression, as detected by immunohistochemistry (n = 6).E Cell proliferation in xenografted tumors of mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression, as detected by Ki-67 staining (n = 6).F Cell apoptosis in xenografted tumors of mice in response to JMJD6 silencing alone or in combination with ERK2 (MAPK1) overexpression, as detected by TUNEL staining (n = 6).*p < 0.05 compared with shCtl + oe-NC-treated mice.#p < 0.05 compared with shJMJD6 + oe-NC-treated mice.Measurement data were represented as mean ± standard deviation.The data comparison between multiple groups was performed by one-way ANOVA with Tukey's post-hoc test

Fig. 7
Fig.7JMJD6 inhibitor SKLB325 represses the LCSC stemness and relieves cell radioresistance.A The expression of HOTAIR, JMJD6 and LSD1 in Hep3B and Huh7 CSCs in response to JMJD6 inhibitor SKLB325 determined by RT-qPCR, normalized to GAPDH.B The expression of HOTAIR and MAPK1 in Hep3B and Huh7 CSCs in response to JMJD6 inhibitor SKLB325 determined by RT-qPCR, normalized to GAPDH.C The expression of Sox2 and Oct2 in Hep3B and Huh7 CSCs in response to JMJD6 inhibitor SKLB325 determined by RT-qPCR, normalized to GAPDH.D The difference of cell stemness after SKLB325 treatment, as detected by microsphere formation assay.E The impact of SKLB325 on colony formation ability in response to SKLB325 treatment, as detected by clonogenic assay.F The diameter of xenografts tumors in mice with SKLB325 treatment.G The tumor volume of xenografted tumors.H, ERK2 (MAPK1) expression in the tumor tissues of mice in response to JMJD6 inhibitor SKLB325 measured by immunohistochemistry.I The expression of HOTAIR in the tumor tissues of mice in response to JMJD6 inhibitor SKLB325 determined by RT-qPCR, normalized to GAPDH.J Ki-67 staining and TUNEL staining were adopted to examine cell proliferation and apoptosis in the tumor tissues of mice in response to JMJD6 inhibitor SKLB325.*p < 0.05 compared with DMSO-treated cells or mice.Experimental data were shown as mean ± standard deviation.Results between two groups were compared by unpaired t test